Chiral 1-(4-methylphenylmethyl)-5-OXO-{N-[3-T-butoxycarbonyl-aminomethyl)]-piperidin-1-yl-pyrrolidine-2-carboxamides as inhibitors of collagen induced platelet activation and adhesion

ABSTRACT

Chiral 1-(4-methylphenylmethyl)-5-oxo-{N-[(3-t-butoxycarbonyl-aminomethyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamides as inhibitors of collagen induced platelet activation and adhesion The present invention provides chiral (2S)-1-(4-methyl-phenylmethyl)-5-oxo-(3S)—{N-[(3-t-butoxycarbonyl aminomethyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamide, and (2S)-1-(4-methylphenylmethyl)-5-oxo-(3R)—{N-[(3-t-butoxycarbonyl amino methyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamide of formula 6 and 7 respectively. The present invention also relates to use of these moieties as inhibitors of collagen induced platelet adhesion and aggregation mediated through collagen receptors. The present invention provides a process for preparation of chiral carboxamides of formula 6 and 7 using the process which has advantage to avoid any racemization at the a-carboxylic center, during N-alkylation. The reagent LiHMDS is used at low temperatures to furnish methyl N-(p methylphenylmethyl)lpyroglutamate in good chiral purity.

FIELD OF THE INVENTION

The present invention relates to chiral1-(4-methylphenylmethyl)-5-oxo-{N-[(3-t-butoxycarbonyl-aminomethyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamides.The present invention also relates to use of these moieties asinhibitors of collagen induced platelet adhesion and aggregationmediated through collagen receptors.

BACKGROUND OF THE INVENTION

Cardiovascular disease (CVD) is a major public health concern. Despitemajor progress in diagnosis and treatment over the years, CVD continuesto represent the most frequent cause of morbidity and mortalityworldwide. Platelets, which are central to normal hemostasis and limitsblood loss after injury, are also the key players in pathologicalconditions, such as deep vein thrombosis and arterial thrombosis. Theypreserve vascular integrity and thereby prevent haemorrhage afterinjury. However, vascular damage, such as rupture of an atheroscleroticplaque, results in a platelet-dependent thrombus formation, which maylead to vascular occlusion with resultant hypoxia and infarction ofdistal tissues. The resulting clinical scenarios encompass stable andunstable angina, acute myocardial infarction (MI), ischaemic stroke andperipheral arterial occlusive disease.

The regulation of platelet-endothelial interaction, and therebyhaemostasis and thrombosis, occurs due to precarious balance betweenactivatory and inhibitory mechanisms that control platelet activationupon exposure to damaged tissues, yet enable platelets to remainquiescent in the undamaged circulation. In healthy vasculature,circulating platelets are maintained in an inactive state by nitricoxide and prostacyclin released by endothelial cells lining the bloodvessels. Endothelial cells also express ADPase (adenosinediphosphatase), which degrades ADP released from red blood cells andactivated platelets, thereby preventing further activation of platelets.When the vessel wall is damaged, the release of these endogenousanti-platelet substances is impaired and subendothelial matrix isexposed. Platelets adhere to exposed collagen and von Willebrand factor(vWF) through receptors that are constitutively expressed on theplatelet surface. Adherent platelets integrate signals from the bindinginteraction, change their shape, secrete ADP from their dense granules,and synthesize and release thromboxane A2 (TXA2). The ADP and TXA2 thatare released serve as platelet agonists by activating ambient plateletsand recruiting them to the site of vascular injury. Disruption of thevessel wall also exposes platelets to collagen and tissuefactor-expressing cells that further trigger the procoagulant response.In an advanced stage of activation, platelets stimulate bloodcoagulation by providing a surface at which the coagulation factors areactivated to generate thrombin. In addition to converting fibrinogen tofibrin, thrombin also serves as a potent platelet agonist and recruitsmore platelets to the site of vascular injury. Activated plateletspotentiate coagulation by expressing phosphatidylserine on theirsurface. When platelets are activated, glycoprotein (GP) IIb/IIIa(αIIbβ3), the most abundant receptor on the platelet surface, undergoesa conformational change, which increases its capacity to bindfibrinogen. Divalent fibrinogen molecules bridge adjacent plateletstogether to form platelet aggregates. Fibrin strands, generated by theaction of thrombin, then weave these aggregates together to form aplatelet-fibrin mesh.

The notion that the targeting of platelet function may be beneficial inthe prevention of thrombosis is borne out by several clinical trials andthe wide use of anti-platelet therapies. Anti-platelet agents can be subclassified, on the basis of their site of action, into those thatinhibit (i) adhesion, (ii) activation, (iii) aggregation, or (iv)platelet-mediated links with inflammation. Of the currently availableagents, aspirin, clopidogrel, dipyridamole, and cilostazol inhibitplatelet activation, albeit via different mechanisms, whereas GPIIb/IIIaantagonists block platelet aggregation. However, there remains asubstantial incidence of arterial thrombosis in patients on currentlyavailable anti-platelet therapy. Limitations of current therapiesinclude weak inhibition of platelet function (for example, by aspirin),blockade of only one pathway of ADP mediated signalling (for example, byclopidogrel), slow onset of action (for example, of clopidogrel),interpatient response variability with poor inhibition of plateletresponse in some patients (for example, to aspirin and clopidogrel), theinability to transform the success of intravenous integrin αIIbβ3antagonist therapy into oral therapy and the inability to completelyseparate a reduction in thrombotic events from an increase in bleedingevents. Therefore, the successes and limitations of current therapiescoupled with the advances made in our understanding of platelet biologyare instructive in the design of new drugs to more effectively regulatevalidated targets.

Despite the complexity of extracellular matrix, platelet collageninteractions play a pivotal role in the initiation of hemostasis andthrombosis in vivo. As stable platelet adhesion and subsequentactivation by collagen is mediated by platelet collagen receptors, theconsequence of inhibition of either of these has gained considerableattention. Furthermore, human studies involving patients lacking eitherreceptor, or equivalent mouse models, reveal reduced plateletresponsiveness to collagen, with only mild deficiencies in haemostasisand do not lead to an increase in spontaneous bleeding tendency.

Thus, inhibition of platelet collagen-receptor activation may representa novel pharmacological target in the search of more selective andspecific antithrombotic agents for the prevention and/or treatment ofacute occlusive arterial thrombosis, eg, myocardial infarction orstroke. Platelet interaction is the first step in the haemostaticprocess, where, extracellular matrix (ECM) is exposed at sites ofinjury. Among the macromolecular constituents of the ECM, collagen isconsidered to play a major role in this process, as in vitro it not onlysupports platelet adhesion through direct and indirect pathways but italso directly activates the cells in initiating aggregation andcoagulant activity. The primary targets of existing anti-platelettherapy are molecules involved in platelet activation and aggregation.At present, there are no drugs in clinical use that block the initialtethering and adhesion of platelets to collagen and von Willebrandfactor and hence their arrest on the blood vessel wall. The inhibitionof this early step in thrombus formation is more likely to reduce orprevent the incidence of arterial thrombosis in patients ofcardiovascular diseases [Review of the subject: PNAS, 2009 vol. 106 no.3 719-724, Small-molecule inhibitors of integrin α2β1 that preventpathological thrombus formation via an allosteric mechanism].

There have been several reports of peptide & proteins which inhibitplatelet aggregation by inhibiting platelet activation by collagen &other endothelial derived activating molecules [FEBS Journal 277 (2010)413-427, Aegyptin displays high-affinity for the von Willebrand factorbinding site (RGQOGVMGF) in collagen and inhibits carotid thrombusformation in vivo; J Thromb Thrombolysis (2007) 24:275-282, Inhibitionof collagen, and thrombin-induced platelet aggregation by Lansberg'shognose pit viper (Porthidium lansbergii hutmanni) venom; ActaBiochimica Polonica, Vol. 50 No. 4/2003, 1119-1128, Inhibition ofcollagen-induced platelet reactivity by DGEA peptide; FEBS Journal 273(2006) 2955-2962, Identification and characterization of acollagen-induced platelet aggregation inhibitor, triplatin, fromsalivary glands of the assassin bug, Triatoma infestans; U.S. Pat. No.5,851,839: Platelet aggregation inhibitors; 6 U.S. Pat. No. 5,756,454,Collagen-induced platelet aggregation inhibitor; U.S. Pat. No.5,710,131, Inhibitor of collagen-stimulated platelet aggregation; U.S.Pat. No. 5,587,360, Platelet adhesion inhibitor].

However there are very few reports of small molecules which are capableof blocking platelet activation/aggregation by collagen & otherendothelial derived activating molecules [(WO/1995/006038) Plateletaggregation inhibitors and references cited therein; U.S. Pat. No.6,221,357, Flavonoids derived from citrus peels as collagen-inducedplatelet aggregation inhibitor; U.S. Pat. No. 5,814,636, Compounds withplatelet aggregation inhibitor activity; U.S. Pat. No. 5,719,145,Amidine derivatives and platelet aggregation inhibitor containing thesame; U.S. Pat. No. 5,698,692, Compound with platelet aggregationinhibitor activity; U.S. Pat. No. 5,629,321, Bicyclic compound andplatelet aggregation inhibitor containing the same; J. Med. Chem. 2007,50, 5457-5462, Small Molecule Inhibitors of Integrin α2β1; PNAS, 2009vol. 106 no. 3 719-724, Small-molecule inhibitors of integrin α2β1 thatprevent pathological thrombus formation via an allosteric mechanism;Cardiovascular Research 75 (2007) 782-792, Characterization of a noveland potent collagen antagonist, caffeic acid phenethyl ester, in humanplatelets: In vitro and in vivo studies; Arterioscler Thromb Vasc Biol2007; 27; 1184-1190; EXP3179 Inhibits Collagen-Dependent PlateletActivation via Glycoprotein Receptor-VI Independent of AT1-ReceptorAntagonism Potential Impact on Atherothrombosis; Phytother. Res. 16,398-399 (2002), Human Platelet Aggregation Inhibitors from Thyme (Thymusvulgaris L.); J. Med. Chem., 2010, 53 (5), pp 2087-2093, StructuralBasis for Platelet Antiaggregation by Angiotensin II Type 1 ReceptorAntagonist Losartan (DuP-753) via Glycoprotein VI]

Amides of N-substituted pyroglutamic acids have been reported asmoderate inhibitor of thrombin (Dikshit et al., 2001 Indian Patent1206/DEL/2001) and has shown anti-thrombotic activity in mice model ofthrombosis.

Therefore, the present invention focuses on the identification andpreparation of pure diastereomers of a class of compounds whichspecifically inhibits collagen mediated platelet aggregation withoutaffecting other regulatory and physiologically relevant plateletfunctions, and thus provides a clinically useful anti-thrombotic agent.The present invention makes pure diastereomers for improvement of theiractivity compared to the mixture of diastereomers.

OBJECTS OF THE INVENTION

The main object of the invention is to provide the compounds whichinhibit collagen induced platelet adhesion and aggregation in in vitroand ex vivo assays. Invention relates to compounds of general formula 6and 7 which are distereomeric forms of chiral1-(4-methylphenylmethyl)-5-oxo-{N-[(3-t-butoxycarbonyl-aminomethyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamidesof general formula I, wherein the general formula 1 comprising thechiral compound of formula 6 and 7.

Another object of the invention is the compounds of formula 6 and 7individually or mixture thereof for preventing thrombosis, plateletadhesion and aggregation and useful in various cardiovascular diseasestates as anti-platelet compound.

Further object of the invention is to provide the compounds havingprolonged duration of action against Collagen-epinephrine inducedthrombosis in comparison to standard drugs such as aspirin andclopidogrel.

Still another object of the invention is to provide the compounds havingsignificantly less prolongation of bleeding time as compared to thestandard anti-platelet drugs such as aspirin and clopidogrel.

One more object of the invention is to provide the compounds havingfaster absorption, higher water solubility and better bio-availability.

Further object provides the process for the preparation of pure forms ofcompound 6 and compound 7.

Furthermore object is to provide compositions for the treatment ofcardiovascular disease comprising the compound of formula 6 or 7individually with pharmaceutically acceptable additives and excipients.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a Chiral1-(4-methylphenylmethyl)-5-oxo-{N-[(3-t-butoxycarbonyl-aminomethyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamidesof general formula 1 wherein the formula 1 comprising the chiralcompound of formula 6 and 7

In an embodiment of the invention the compounds are useful forpreventing thrombosis, platelet adhesion and aggregation.

The preferred embodiment of this invention provides the compound offormula 6 and 7 individually or mixture thereof for preventingthrombosis, platelet adhesion and aggregation and useful in variouscardiovascular disease states as anti-platelet compound.

More preferred embodiment of this invention provides compound of formula6 is more active with long lasting effect when compared against standarddrug Aspirin and Clopidogrel as well as compound 1 and 7 inCollagen-epinephrine induced pulmonary thromboembolism in mice.

Another embodiment of this invention provides the compound 1, 6 and 7(30 μM/kg, 1 hr p.o. dosing) exhibited a prolongation in bleeding time,which was significantly less as compared to the standard anti-plateletdrug, aspirin and clopidogrel.

Yet another embodiment provides the compound 1, 6 and 7 are activeagainst collagen induced aggregation in vitro in human platelet richplasma while no effect on ADP induced platelet aggregation, and compound6 and 7 are more specific than compound 1 which exhibits moderateefficacy against thrombin mimetic, SFLLRN (TRAP) induced plateletaggregation.

Yet another preferred embodiment provides compound 6 is better thancompound 7 in attenuating collagen stimulated platelet aggregation inhyperlipidemic hamsters.

Another embodiment is the compound 6 exhibiting dose dependent reductionin platelet adhesion over collagen matrix and comparatively better thanaspirin in mice (ex vivo).

Another embodiment is that the compound 6 is better than compound 7 ininhibiting platelet adhesion over, collagen coated surface GPVI- andα2β1-mediated platelet adhesion assay on collagen (Human, in-vitro).

In another embodiment it is discussed that in hyperlipidemic hamsters,the compound 6 exhibits significant attenuation in platelet adhesionover collagen surface in contrast to compound 7 which exhibits no effecton the same.

Further embodiment provides the compound 1 and 7 non-specificallyattenuated thrombin amidolytic activity while compound 6 did not exhibitany of such non-specific thrombin inhibitory property.

Furthermore embodiment provides the compound 6 significantly reducesthrombus weight even at 10 μM/kg in arterio-venous shunt model in ratand hyperlipidemic hamsters.

Even further embodiment provides the compound 6 significantly prolongedthe time to occlude the carotid artery in rats (FeCl₃ induced arterialthrombosis), thus displaying its remarkable antithrombotic potential,while compound 1 failed to exhibit any protection at the same dose.

Even furthermore embodiment provides the compound 6 significantlyincreased the time to occlusion in hyperlipidemic hamsters, thusconfirming its substantial antithrombotic efficacy in a disease model,while compound 7 remained ineffective and thus did not displayantithrombotic characteristic in the same.

Further embodiment provides that the compound 6 exhibits fasterabsorption and prolonged systemic exposure for more than 24 hrs {C_(max)(149.49±53.12), t_(max) 0.75±0.144 hrs} and higher water solubility(416.41±62.35 μg/ml) when compared to compound 7 {C_(max)(112.81±169.77,t_(max) 8.67±0.66)} and water solubility (71.75±13.45 μg/ml); and thecompound 1 comparatively exhibits fast absorption leading to C_(max) of947.02±237.4 ng/ml.

Another embodiment provides the process for the preparation of compound6 and compound 7 wherein the process steps comprising of;

-   reacting methylpyroglutamate with p-methyl benzyl bromide in an    aprotic solvent in presence of LiHMDS at a temperature ranging    between 0 to 35° C. for a period ranging between 1 to 4 hr,    quenching the reaction mixture with HCl and obtaining the compound    of formula 2,

-   reacting compound of formula 2 with sodium carbonate at a    temperature ranging between 0° C. to 30° C. for a period ranging    between 0.5 to 4 hr to obtain    (2S)—N-(p-methylphenylmethyl)pyroglutamic acid of formula 3

-   reacting compound of formula 3 with compound of formula    4[(3R)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine] or    5[(3S)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine] in an aprotic    solvent selected from a group consisting of dichloromethane,    tetrahydrofuran and dioxane, in presence of a coupling reagent    selected from the group consisting of dicyclohexylcarbodiimide and    benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium    hexafluorophosphate, OR an activating agent 1-hydroxy benzotrizole    or isobutyl chloroformate at a temperature ranging between −20° C.    to +30° C. for a period ranging from 1 to 3 hrs followed by    purification using chromatography to produce compound of formula 6    or 7 respectively.

In an embodiment of the invention wherein the compound of formula 3 isreacted with oxalyl chloride at 0° C. to obtain the acid chloridefollowed by reaction with compound of formula 4 or 5 in presence oftriethylamine in dichloromethane at room temperature for a periodranging from 2 h to 3 h to obtain the compound of formula 6 or 7respectively.

In another embodiment of the invention wherein the compound of formula 3is reacted with compound of formula 4 or 5 in presence of a couplingreagent dicyclohexylcarbodiimide and 1-hydroxy benzotrizole indichloromethane at a temperature ranging between −5° C. to 0° C. for aperiod ranging between 2 h to 3 h to obtain compound of formula 6 or 7respectively.

In a further embodiment of the invention wherein the compound of formula3 is reacted with compound of formula 4 or 5 in the presence ofdiisopropyl ethylamine, andbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, in dichloromethane at temperature ranging between −5° C. to00° C. for a period ranging between 2 h to 3 h followed by stirring attemperature at temperature ranging between 0° C. to 25° C. to obtaincompound of formula 6 or 7 respectively.

In still another embodiment of the invention wherein the compound offormula 3 is reacted with compound of formula 4 or 5 in presence oftriethyl amine, and isobutyl chlorormate, in THF at −20° C., for aperiod ranging between 1 h to 2 h followed by stirring at temperature attemperature ranging between 0° C. to 25° C. to obtain the compound offormula 6 or 7.

Even furthermore embodiment provides the purification of compound 6 and7 is carried out by crystallization using the solvents selected from agroup consisting of pentane, hexane, cyclohexane, toluene, ethylacetate.

Another embodiment provides the use of said compounds 6 or 7 fortreatment of coronary syndrome (ACS) such as ST-segment elevationmyocardial infarction (MI), non-ST segment elevation MI, unstableangina, thrombotic stroke and in patients of angioplasty to preventplatelet activation and adhesion.

Furthermore embodiment provides compounds for the treatment ofcardiovascular disease comprising the compound of formula 6 or 7individually with pharmaceutically acceptable additives and excipients.

Furthermore embodiment provides composition wherein the pharmaceuticallyacceptable additive is selected form a group consisting of DMSO, gumacacia or CMC, beta cyclodextrin, or any other pharmaceuticallyacceptable excipients.

BRIEF DESCRIPTION OF DRAWINGS

(*n=no. of experiments)

FIG. 1: Effect of compound 1, 6 and 7 on collagen-epinephrine inducedpulmonary thromboembolism in mice in a time dependent study. Results areexpressed as Mean±SEM (n=5, 10 animals/group/experiment)

FIG. 2: Effect of compound 1, 6 and 7 on tail bleeding time in mice in atime dependent study Results are expressed as Mean±SEM (n=5, 10animals/group/experiment).

FIG. 3 a: Effect of compounds 6 and compound 7 on aggregation of humanplatelet rich plasma (in vitro) induced by (i) Collagen (ii) Convulxinand Ristocetin (iii) ADP and TRAP. Results are expressed as Mean±SEM(n=3)

FIG. 3 b: Effect of compound 6 on aggregation of mice platelet richplasma (ex vivo) induced by Collagen, ADP and Thrombin. Results areexpressed as Mean±SEM (n=5, 5 animals/group/experiment).

FIG. 3 c: Whole blood aggregation responses of chow fed and highcholesterol high fat fed hamsters induced by ADP (10 μM), Collagen (2.5μg/ml) and Thrombin (1 U/ml) (no. of animals=10/group). (i) effect ofstandard anti-platelet drugs and compounds 6 & 7 on whole bloodaggregation (ii) effect of compound 6 on platelet aggregation in dosedependent manner (n=10, no. of animals=10/group). Results are expressedas Mean±SEM.

FIG. 4 a: Effect of compounds 6 & 7 on human platelet adhesion on (a)fibrillar and (b) soluble collagen coated surface. Results are expressedas Mean±SEM (n=3).

FIG. 4 b: Adhesion of mouse platelets on collagen matrix as observedunder fluorescence microscope 40× (ex vivo method). Different picturesshow the different treatment performed. Washed murine platelets(2×10⁶/ml) were obtained from animals pretreated with the compound(compound 6) (different doses) and seeded on BSA or Collagen (fibrillar)coated cover slips (A, B). Platelets from untreated animals were allowedto adhere on uncoated and BSA coated cover slip respectively todelineate the possibility of non specific binding. C, D. controlplatelets were adhered on collagen coated cover slip (control). E, F&G.Platelets obtained from animals pre-treated with compound 6 (10 μM/kg,30 μM/kg & 100 μM/kg) respectively were adhered on collagen coated coverslip. H, I. Platelets from Aspirin (100 μM/kg) & Clopidogrel treated(100 μM/kg) animals resp. (standard Drugs) and adhered on collagencoated cover slip (n=5, 5 animals/group/experiment).

FIG. 4 c) (i) Effect of compound 6 & 7 and standard drugs on plateletadhesion (ex-vivo) in hyperlipidemic hamsters on collagen coated surface(ii) effect of compound 6 on platelet adhesion in a dose dependent way.Results are expressed as Mean±SEM (no. of animals=10/group).

FIG. 5: Effect of compound 1, 6 and 7 on amidolytic activity of humanalpha-thrombin using fluorogenic substrate. Results are expressed asMean±SEM.

FIG. 6 a: (i) Effect of compounds 6 and 7 in reducing the thrombusweight and their efficacy was compared to those of standardanti-platelet drugs, aspirin and clopidogrel in rats. (ii) Effect ofcompounds 6 in dose dependent manner. Results are expressed as Mean±SEM(n=3)

FIG. 6( b): Effect of compound 6 on High fat diet treatment whichincreased the thrombus weight, thus displaying a pro-thrombotic stateinduced by hyperlipidemic diet mimicking the clinical condition. Resultsare expressed as Mean±SEM (n=3).

FIG. 7 a) (i) Effect of compound 6 & 7 on total time to occlusion (TTO)in ferric chloride induced arterial thrombosis in rats (n=6) (ii) Effectof compound 6 on time to occlusion (TTO) in rats in a dose dependentway. Results are expressed as Mean±SEM (no. animals=6/group).

FIG. 7 b: Effect of compound 6 & 7 on total time to occlusion (TTO) inferric chloride induced arterial thrombosis in hyperlipidemic hamsters.Results are expressed as Mean±SEM (no. of animals=6/group)

FIG. 8: Effect of compound 6 on intracellular calcium mobilization inFura-2 loaded human platelets stimulated by collagen (5 μg/ml) (n=3)

FIG. 9: Effect of compound 6 on tyrosine phosphorylation of plateletproteins after stimulation with collagen (2 μg/ml) (n=3)

FIG. 10: Plasma concentration time profile of compound 1, 6 & 7 (n=3)

ABBREVIATIONS

ADP, adenosine diphosphate, PMA, 12-phorbol 13-myristate acetate, AA,Arachidonic Acid, TRAP, thrombin receptor activated peptide, EGTA,ethylene glycol tetra acetic acid, ACD, acid-citrate-dextrose, gp,glycoprotein, PBS, phosphate buffered saline, FeCl₃, ferric chloride,TTO, time to occlusion, HRP, horseradish peroxidise, CMC, carboxymethylcellulose, PRP, platelet rich plasma, PPP, platelet poor plasma, DMSO,dimethyl sulfoxide, HEPES,(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), SDS, sodiumdodecyl sulphate, PMSF, phenyl methyl sulphonyl fluoride, TBST, trisbuffered saline with Tween-20, p-Tyr, phospho-tyrosine BSA, bovine serumalbumin, HCHF, high cholesterol high fat, SNP, Sodium Nitroprusside, μg,microgram, μM, micromolar, kDa, kilo Dalton, C_(max), Maximum plasmaconcentration, tmax, time to achieve maximum plasma concentration, AUC,Area under curve, MRT, mean residence time

LiHMDS, Lithium bis-(trymethylsilyl)amide, DIC, N,N′-Diisopropylcarbodiimide, HOBt, 1-Hydroxybenzotriazole, PyBOP,Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, DCM,Dichloromethane, HPLC, High Performance Liquid Chromatography, IR,Infrared, NMR, Nuclear Magnetic Resonance spectoscopy, FAB MS, Fast AtomBombardment Mass Spectrocopy, Na₂SO₄, Sodium sulphate, HCl, Hydrochloricacid, MP, melting point, CDCl₃, duteriated chloroform, MHz, Mega hertz,mmol, millimol, [α]_(D), Specific rotation, 6, Chemical shift, KBr,Potassium bromide

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, this invention provides compounds which inhibit collageninduced platelet adhesion and aggregation in in vitro assays and ex vivocollagen induced comparative platelet adhesion assays represented byCompound 1 which as stated in the Indian patent (1206/DEL/2001) is a 1:1mixture of diastereomers Compound 6 and Compound 7, which could not beseparated by crystallization, or by chromotagraphic methods such asHPLC, more particularly, but not to be construed as limiting, thepresently disclosed and claimed invention relate to the chiral synthesisof both diastereomers and the separation of biological actions in thepreferred compound (Compound 1) so as to identify one of the chirallypure diastereomers (compound 6) as having superior anti-thromboticefficacy.

The invention efficiently utilizes the tartarate salts for the chiralresolution of racemic Piperidin-3-ylmethyl-carbamic acid tert-butylester, similar to reported procedure (WO2006/28904 A1, 2006) to obtain3S or 3R piperidin-3-ylmethyl-carbamic acid tert-butyl ester.

N-p-methyl-phenylmethyllpyroglutamic acid was prepared according to theliterature reported protocol described in the experimental section.

In an inventive step, to avoid any racemization at the α-carboxyliccenter, during N-alkylation, the reagent LiHMDS at low temperatures isclaimed to furnish Methyl N-p-methyl-phenylmethyllpyroglutamate in goodchiral purity.

To avoid any racemization at the α-carboxylic center, for example,during coupling process, very efficient and mechanism specific couplingagents are generally used as DCC with HOBt or PyBOP in dry DCM, but thismay not preclude the use of simple acid chloride mediated couplings, asthe invention provides compounds in microcrystalline form and repeatedcrystallization may afford these compounds in almost 99% chiral purity(chiral HPLC), as otherwise indicated.

Asprin and clopidogrel are used as standard drugs and gum acacia, CMC asvehicles.

The invention utilizes a mouse model of collagen-epinephrine inducedpulmonary thrombombolism and bleeding time to justify the effectiveness(for a extended period) of compound 6 over its analog compound 7 andtheir diastereomeric mixture, compound 1 (FIG. 1, 2).

The invention discusses the studies detailing theanti-adhesive/anti-aggregating activities of two compounds (6 & 7) invarious in-vitro/ex-vivo/in-vivo models manifesting betterpharmacological and pharmacokinetic profile over the diastereomericmixture, compound 1.

The invention includes compound 1, 6 and 7 to be active against collageninduced aggregation in vitro in human platelet rich plasma. The in vitroanti-aggregatory activity was comparable among all the three compoundsas they inhibited collagen mediated effects at similar concentrations(FIG. 3 a).

Further, the invention includes compounds 6 and 7 not to exhibit anyeffect on ADP, Ristocetin and thrombin mimetic, SFLLRN (TRAP) inducedplatelet aggregation, while the diastereomeric mixture, Compound 1,exhibited a significant inhibition against TRAP induced plateletaggregation, thereby displaying its non-specific mode of action (FIG. 3b, c).

The invention includes compound 6, even at higher concentrations (500μM) does not significantly inhibit platelet aggregation induced byconvulxin, a GP VI receptor agonist but inhibits collagen inducedaggregation (FIG. 3 a).

Another preferred embodiment includes compound 6 exhibits a dosedependent reduction in collagen mediated platelet aggregation in micewhen evaluated ex vivo (FIG. 3 b).

Further the invention discusses measuring aspirin mediated plateletaggregation inhibition in HCHF fed Hamsters wherein aspirin can onlyinhibit ADP and Thrombin but not collagen induced aggregation. (FIG. 3c)

Measuring clopidogrel mediated platelet aggregation inhibition in HCHFfed Hamsters wherein the clopidogrel can only inhibit ADP inducedaggregation. (FIG. 3 c)

Further measuring compound 1 and 6 mediated platelet aggregationinhibition in HCHF fed Hamsters while compound 7 remained ineffectiveand the preferred compound (Compound 6) is claimed to significantlyinhibit collagen induced platelet aggregation in high fat fed groups ina dose dependent manner (FIG. 3 c).

Compound 1, 6 and 7 partially inhibited both GP VI and GP Ia IIamediated human platelet adhesion over collagen (in vitro) while theeffect was more prominent in GP VI mediated pathway (FIG. 4 a).

Treatment with preferred compound 6 is claimed to significantly reducemice platelet adhesion on collagen coated plates which are comparable toclopidogrel treatment (FIG. 4 b).

Measurement of the platelet hyperactivity in HCHF fed hamsters throughisolated platelet adhesion assays on both collagen and fibrinogen coatedplates. The invention, Compound 1 and 6, exhibit significant reductionin platelet adhesion which are comparable to clopidogrel treatment,whilst treatment with Compound 7 and Aspirin are found insignificant inreducing platelet adhesion on the collagen coated surface inhyperlipidemic hamster model of atherosclerosis (FIG. 8 a). Thepreferred compound 6 exhibits a dose dependent reduction in diet inducedplatelet hyperactivation when adhered over collagen coated surface inhyperlipidemic hamsters (FIG. 4 c).

The invention, in a set of experiments describes the effect of Compound1, 6 and 7 on amidolytic activity of human α-thrombin using fluorogenicsubstrate was evaluated where unlike compound 1 and compound 7, compound6 showed negligible activity. (FIG. 5)

The invention, in general, utilizes several in vivo & ex-vivo assays toascertain their antithrombotic potency in AV-shunt model, Ferricchloride induced carotid thrombosis, to justify the efficacy of 6 over 7against various thrombosis models. [FIG. 6 a,b, FIG. 7 a,b]

The invention, in another set of experiments finds that the preferredcompound (compound 6) inhibits collagen induced increase in [Ca²⁺]i andalso inhibition of tyrosine phosphorylated levels of few (unexplored)proteins that involve in collagen bound GP-VI mediated intracellularsignaling is worked out in this invention. (FIG. 8, 9)

The invention comprehends platelet collagen receptors GP-VI as theplausible target for the preferred compounds but put no remarks onspecific receptor subtype.

The invention discloses the plasma pharmacokinetic profile of 6 over 7,the two promising compounds of the present invention; reveal goodsystemic availability in experimental animals correlating well with itspharmacodynamic properties. (FIG. 10)

The invention is further illustrated by the following examples whichshould not, however, be construed to limit the scope of the presentinvention.

Examples 1 Methyl 2S—N-(p-methylphenyl methyl)pyroglutamate (Compound 2)

Methyl pyroglutamate (10 g, 1 eq, 70 mmol) was dissolved in dry THF andcooled to −30° C., fitted with a septum under N₂ atmosphere. LiHMDS(83.83 ml, 1.2 eq, 77 mmol) was added dropwise and stirred at thistemperature for 45 min. p-methyl benzyl bromide (14.2 g, 1.1 eq, 76mmol), dissolved in dry THF added to the reaction mixture drop wise andstirred from 0° C. to room temperature for 4 hrs. The reaction mixturewas quenched with cold 1N hydrochloric acid and extracted with ethylacetate. The organic layer was separated and dried on Na₂SO₄ andconcentrated. The ester was purified by flash chromatography (silicagel, 230-400 mesh using chloroform as solvent system) to furnishcompound 2 which was used directly in the next step.

Examples 2 Synthesis of (2S)—N-(p-methylphenyl methyl)pyroglutamic acid(Compound 3)

Compound 2 (6.0 g) was then dissolved in methanol (25 ml) and cooled to0° C. 20% sodium carbonate solution (75 ml) was then added to thereaction mixture portion wise. The reaction mixture was then stirred atroom temperature for 4 hours. Methanol was then distilled off and thereduced reaction mixture was then extracted with ether (1×25 ml). Theaqueous layer was acidified with conc. HCl and extracted with ethylacetate (3×30 ml). The organic layer was dried and concentrated to giveoily product (5.2 g) which solidified on standing and was crystallizedfrom hot ethyl acetate.

Yield: 6.9 g, 42% (crystallized product)

[α]_(D) ^(27° C.): +33.96 (c=0.10; Methanol)

M.P.: 86-88° C.

IR (KBr)

-   -   3758, 3452, 2962, 1969, 1663, 1453, 1422, 1281, 1024, 801 cm⁻¹

¹H NMR (CDCl₃, 200 MHz)

2.05-2.18 (m, 1H, 3-H_(a)); 2.20-2.27 (m, 1H, 3-H_(b)); 2.32 (s, 3H,—CH₃); 2.50-2.60 (m, 2H, 4-H); 3.88 (d, 1H, —NCHPh); 4.02-4.04 (m, 1H,2-H); 5.09-5.17 (d, 1H, —NCHPh); 7.12 (s, 4H, Ph-H)

¹³C NMR (CDCl₃, 200 MHz)

14.57, 21.52, 23.26, 30.10, 45.83, 59.01, 61.00, 128.95, 129.92, 132.62,138.11, 174.74, 176.90

FAB MS (m/z):

-   -   234 (M+1)⁺

Example 3 Synthesis of(3R)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine (compound 4)

N-(t-butoxycarbonyl)-3-aminomethyl piperidine (10 g, 1 eq, 47 mmol),(+)-O,O′-Di-p-tolouyl-D-tartaric acid (15.52, 1 eq, 47 mmol) and drymethanol (100 ml) were mixed and heated slowly to reflux just to get asolution. This solution was cooled to room temperature and stirred atthis temperature for 5-6 hours to give a white suspension of the saltwhich was filtered and washed with minimum quantity of dry methanol. Thecrude salt was crystallized once from methanol and the salt thusobtained was suspended in distilled water (25 ml) and cooled to 0° C.10% solution of sodium carbonate solution (100 ml) was then addedportion wise till the suspension was strongly basic. The reactionmixture stirred for additional 10 minutes and was extracted with ethylacetate (5×50 ml). The combined organic layers were dried (Na₂SO₄) andconcentrated under reduced pressure to obtain the compound of formula 4.

Yield: 3.2 g, 62%

-   -   [α]_(D) ^(27° C.): −8.97 (c=1.0; Methanol)

MP: 64-66° C.

IR (Neat):

-   -   3362, 2970, 1703, 1520, 1454, 1365, 1256, 1172 cm⁻¹

¹H NMR (CDCl₃, 300 MHz)

δ 1.00-1.22 (m, 2H, H-4); 1.40 (s, 9H, —O—C(CH₃)₃); 1.57-1.72 (m, 3H,H-3, H-5); 2.21-2.32 (m, 1H, CH_(a)NHBoc); 2.44-2.2.57 (m, 1H,CH_(b)NHBoc); 2.92-3.05 (m, 4H, H-2, H-4); 4.72 (brs, 1H, NH)

¹³C NMR (CDCl₃, 300 MHz)

-   -   26.56, 29.03, 29.58, 38.44, 44.99, 47.46, 51.16, 79.73, 156.72

FAB MS (m/z):

-   -   215 [M+1]⁺, 114

Example 4 Synthesis of(3S)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine (compound 5)

N-(t-butoxycarbonyl)-3-aminomethyl piperidine (10 g, 1 eq, 47 mmol),(−)-O, O′-Di-p-tolouyl-L-tartaric acid (15.52, 1 eq, 47 mmol) and drymethanol (100 ml) were mixed and heated slowly to refluxing to just toget a uniform solution. The reaction mixture was cooled to roomtemperature and stirred at this temperature for 5-6 hours. White solidformed was filtered out and washed with minimum quantity of drymethanol. The crude solid was recrystallized from methanol. The compoundwas suspended in distilled water (25 ml) and cooed to 0° C. 10% solutionof sodium carbonate solution (100 ml) was then added portion wise, untilbasic, with stirring for 10 min. The reaction mixture was extracted withethyl acetate (5×50 ml). The organic layer was separated, dried andconcentrated under reduced pressure to obtain the compound of formula 3.

Yield: 3.28 g, 65%

[α]_(D) ^(27° C.): +11.03 (c=0.10; Methanol)

MP: 64-66° C.

IR (Neat):

-   -   3360, 2972, 1703, 1519, 1455, 1365, 1255, 1172 cm⁻¹

¹H NMR (CDCl₃, 300 MHz) δ 1.01-1.21 (m, 2H, H-4); 1.39 (s, 9H,—O—C(CH₃)₃); 1.57-1.72 (m, 3H, H-3, H-5); 2.20-2.31 (m, 1H,CH_(a)NHBoc); 2.49-2.56 (m, 1H, CH_(b)NHBoc); 2.90-3.03 (m, 4H, H-2,H-4); 4.77 (brs, 1H, NH)

¹³C NMR (CDCl₃, 300 MHz)

-   -   26.56, 29.03, 29.58, 38.44, 44.99, 47.46, 51.16, 79.73, 156.72

FAB MS (m/z):

-   -   215 (M+1)⁺, 114

Example 5 Synthesis of(2S)-1-(4-methylphenylmethyl)-5-oxo-(3S)—{N-[(3-t-butoxycarbonylaminomethyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamide, (compound 6)

Method A

Step-1: (2S)—N-(4-methylphenylmethyl)-pyroglutamic acid, Compound 3(3.50 g, 1 eq, 15 mmol) was dissolved in dry DCM (50 ml) and cooled to0° C. Oxalyl chloride (1.3 ml, 1.2 eq, 18 mmol) was then added drop wiseat 0° C. and the mixture was allowed to warm up to room temperature andstirred for 12 hours. The reaction mixture was then concentrated overreduced pressure.

Step-2: Compound 4 (3.53 g, 1.1 eq., 16 mmol) was dissolved in dry DCM(50 ml) and cooled to 0° C. Triethylamine (4.82 ml, 2.3 eq, 35 mmol) wasthen added to the reaction mixture and stirred for 10 min. The acidchloride (from step-1) dissolved in dry DCM (25 ml) was then added dropwise at 0° C. and stirred at room temperature for 3 hrs. The reactionmixture was then washed with saturated sodium bicarbonate (1×50 ml), icecold 1N HCl (1×50 ml) and then with brine. The organic layer wasseparated, dried and concentrated. The viscous oil obtained was thenflash chromatographed [silica gel, 230-400 mesh usingchloroform-methanol (9.5:0.5) as solvent system] to get pure compound.The compound was further purified by re-crystallization from ethylacetate-hexane.

Yield: 4.25 g, 66% (double crystallized)

[α]_(D) ^(27° C.): +35.00 (c=0.098; Chloroform)

MP: 138-140° C.

IR (KBr):

3274, 2977, 2934, 2860, 1714, 1674, 1630, 1541, 1449, 1365, 1272, 12489,1176, 1144, 1039, 997, 958, 847, 759, 717, 654

¹H NMR (CDCl₃; 600 MHz):

δ 1.119-1.390 (m, 2H, 4′-H); 1.448 (s, 9H, —O—C(CH₃)₃); 1.704 (m, 1H,3′-H); 1.725-1.887 (m, 2H, 5′-H); 2.138-2.175 (m, 1H, 3-H_(a)); 2.305(s, 3H, Ph-CH3), 2.361-2.398 (m, 1H, 3-H_(b)); 2.496-2.555 (m, 2H, 4-H);2.646-2.757 (m, 2H, —CH₂—NH-Boc); 2.833-3.090 (m, 2H, 6′-H); 3.324-3.428(m, 1H, 2′-H_(a)); 3.731-3.769 (m, 1H, 2′-H_(b)); 4.110-4.200 (m, 2H,—CH₂-Ph & 2-H); 4.871 (s, 1H. NH); 5.133-5.138 (d, 1H, —CH₂-Ph);7.060-7.090 (dd, 4H, Ph-H)

¹³C NMR (CDCl₃; 600 MHz):

21.063, 21.086, 22.651, 24.614, 27.898, 28.287, 28.639, 29.748, 35.955,38.330, 42.728, 43.118, 43.332, 45.065, 45.646, 49.273, 55.673, 55.841,79.331, 128.427, 128, 733, 129.351, 133.078, 137.339, 156.028, 168.810,169.253, 175.164

FAB MS

-   -   (m/z): 429 (M⁺), 330, 374, 452 (M+Na)⁺

Method-B

(2S)—N-(4-methylphenylmethyl)-pyroglutamic acid, Compound 3 (3.50 g, 1eq, 15 mmol) and 1-hydroxy benzotrizole (3.04 g, 1.5 eq, 22.5 mmol) weredissolved in dry DCM (50 ml) in a three necked round bottom flask fittedwith N₂ inlet. The reaction mixture was cooled to 0° C. in an ice-saltbath. Dicyclohexylcarbodiimide (DCC) (3.72 g, 1.2 eq, 18 mmol) dissolvedin dry DCM (20 ml) was added to it and stirred while being at 0° C. for15 min. (3R)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine, Compound 4(3.21 g, 1 eq, 15 mmol) dissolved in dry DCM (20 ml) was added drop wiseto the reaction mixture and stirring was continued for 3 hrs at 0° C.The reaction mixture was the brought to room temperature andconcentrated under reduced pressure. The residue was dissolved in ethylacetate, cooled to 5° C., filtered and the filtrate was washedsuccessively with dil. citric acid (3×50 ml), dil. sodium bicarbonatesolution (3×50 ml) and brine (1×50 ml). The organic layer was separated,dried and concentrated under reduced pressure. The crude material wasflash chromatographed [silica gel, 230-400 mesh usingchloroform-methanol (9.5:0.5) as solvent system] to get pure Compound 6,4.5 g, 70% yield.

Method-C

(2S)—N-(4-methylphenylmethyl)-pyroglutamic acid acid, Compound 3 (3.5 g,1 eq, 15 mmol), (3R)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidineCompound 4 (3.21 g, 1 eq, 15 mmol) and diisopropyl ethyl amine (5.36 ml,2 eq, 30 mmol) were dissolved in dry DCM (50 ml) in a three necked roundbottom flask fitted with N₂ inlet and rubber septa. The reaction mixturewas cooled to 0° C. in an ice-salt bath and stirred for 10 min. Asolution of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (7.82 g, 1 eq, 15 mmol) dissolved in dry DCM (20 ml)was added drop wise to the reaction mixture and stirring was continuedfor 3 hours at 0° C. The reaction mixture was then brought to roomtemperature and concentrated under reduced pressure. The residue wasdissolved in ethyl acetate, washed successively with dil. citric acid(3×50 ml), dil. sodium bicarbonate solution (3×50 ml) and brine (1×50ml). The organic layer was separated, dried and concentrated underreduced pressure. The crude material was flash chromatographed [silicagel, 230-400 mesh using chloroform-methanol (9.5:0.5) as solvent system]to get pure compound 4.6 g, 72% yield.

Method-D

(2S)—N-(4-methylphenylmethyl)-pyroglutamic acid Compound 3 (3.5 gm, 1eq, 15 mmol) and dry triethyl amine (4.10 ml, 2 eq, 30 mmol) weredissolved in dry THF (50 ml) in a three necked round bottom flask fittedwith dry N₂ inlet and rubber septa. The reaction mixture was cooled to−20° C. and stirred while being at −20° C. for 10 minutes. Isobutylchloroformate (1.96 ml, 1 eq, 15 mmol) was added drop wise to it at −20°C. and the stirring was continued for 15 minutes. Solution of(3R)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine, Compound 4 (3.21 gm,1 eq, 15 mmol) in dry dichloromethane (25 ml) was added drop wise to itin 10 minutes at −20° C. The reaction mixture was stirred for 1 hr at 0°C. to 25° C., quenched by adding saturated NH₄Cl solution andconcentrated under reduced pressure. The residue was dissolved in ethylacetate and washed successively with dil. citric acid (3×50 ml), dilsodium bicarbonate solution (3×50 ml) and brine (1×50 ml). The organiclayer was separated, dried over Na₂SO₄ and concentrated under reducedpressure. The crude material was flash chromatographed [silica gel,230-400 mesh using chloroform-methanol (9.5:0.5) as solvent system] toget pure Compound 6 4.2 g, 65% yield.

Example-6 Synthesis of(2S)-1-(4-methylphenylmethyl)-5-oxo-(3R)—{N-[(3-t-butoxycarbonyl-aminomethyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamide(compound 7)

Method A

Step-1: (2S)—N-(4-methylphenylmethyl)-pyroglutamic acid, (3.50 g, 1 eq,15 mmol) was dissolved in dry DCM (10 ml) and cooled to 0° C. Oxalylchloride (1.3 ml, 1.2 eq, 18 mmol) was then added drop wise at 0° C. andthe mixture was allowed to warm up to room temperature and stirred for12 hours. The reaction mixture was then concentrated over reducedpressure.

Step-2: Compound 5 (3.53 g, 1.1 eq., 16 mmol) was dissolved in dry DCM(20 ml) and cooled to 0° C. Triethylamine (4.82 ml, 2.3 eq., 35 mmol)was then added to the reaction mixture and stirred for 10 min. The acidchloride (from step-1) dissolved in dry DCM (10 ml) was then added dropwise at 0° C. and stirred at room temperature for 3 hrs. The reactionmixture was then washed with saturated sodium bicarbonate (1×50 ml), icecold 1N HCl (1×50 ml) and then with brine. The organic layer wasseparated, dried and concentrated. The viscous oil obtained was thenflash chromatographed [silica gel, 230-400 mesh usingchloroform-methanol (9.5:0.5) as solvent system] to give the compoundwhich was further purified by re-crystallization from ethylacetate-hexane to give pure compound, 4.38 g, 68% yield (doublecrystallized)

[α]_(D) ^(27° C.): +5.69 (c=0.99; Chloroform)

MP: 180-182° C.

IR (KBr):

3330, 2974, 2935, 2857, 1710, 1677, 1642, 1528, 1444, 1363, 1274, 1173,1146, 1080, 997, 958, 850, 756, 645

¹H NMR (CDCl₃; 600 MHz):

δ 1.177-1.270 (m, 2H, 4′-H); 1.424 (s, 9H, —O—C(CH₃)₃); 1.668 (m, 1H,3′-H); 1.768-1.856 (m, 2H, 5′-H); 2.138-2.175 (m, 1H, 3-H_(a)); 2.305(s, 3H, Ph-CH₃), 2.361-2.398 (m, 1H, 3-H_(b)); 2.496-2.555 (m, 2H, 4-H);2.646-2.757 (m, 2H, —CH₂—NH-Boc); 2.833-2.984 (m, 2H, 6′-H); 3.324-3.428(dd, 1H, 2′-H_(a)); 3.731-3.769 (m, 1H, 2′-H_(b)); 4.160-4.180 (m, 2H,—CH₂-Ph & 2-H); 4.871 (s, 1H. NH); 5.133-5.138 (m, 1H, —CH₂-Ph);7.060-7.090 (dd, 4H, Ph-H);

¹³C NMR (CDCl₃; 600 MHz):

21.086, 22.728, 23.866, 24.614, 28.096, 28.303, 28.700, 29.784, 36.405,37.322, 42.828, 43.011, 43.263, 45.042, 45.852, 48.441, 55.986, 56.047,79.316, 128.389, 128.710, 129.336, 133.048, 137.339, 156.080, 168.810,169.001, 175.072

FAB MS

-   -   (m/z): 429 (M⁺), 330, 374, 452 (M+Na)⁺

Biological Study

In Vitro Studies

From human subjects blood was collected in citrate-phosphate-dextrose(CPD) (1:7) from healthy volunteers (age between 18-60 years) afterprior consent. A detailed medical history and physical examination wascarried out before phlebotomy. The donors were free from heart, lung,kidney disease, cancer, epilepsy, diabetes, tuberculosis, abnormalbleeding tendency, allergic disease, sexually transmitted diseases,jaundice, malaria, typhoid and thyroid or any other endocrine disorder.Donors were free from any prior medication for last 72 hours.

Preparation of Washed Platelets

Fresh blood was drawn by venipuncture from consenting healthy humanvolunteers in citrate-phosphate-dextrose. Platelet-rich plasma (PRP) wasobtained by centrifugation at 180 g for 20 minutes at room temperature(Beckman T J6, USA). 10% (v/v) of ACD buffer (39 mM citric acid, 75 mMtri-sodium citrate.2H₂O, 135 mM D-glucose, pH 4.5) was added, and theplatelet-rich plasma was spun at 800 g for 10 minutes (3K30 SigmaCentrifuge, Germany). Platelets were then washed twice with Buffer (20mM HEPES, 138 mM NaCl, 2.9 mM KCl, 1 mM MgCl₂, 0.36 mM NaH₂PO₄, 1 mMEGTA, 4.77 mM trisodium citrate, and 2.35 mM citric acid, 5 mM glucoseand Apyrase 1 U/ml, pH 6.5) containing 0.1% bovine serum albumin (BSA)and finally resuspended in the HEPES-buffered Tyrode solution (pH 7.4),to give a concentration of 2×10⁸ cells/mL (Jandrot-Perrus M, Lagrue A H,Okuma M, and Boni C Adhesion and Activation of Human Platelets Inducedby Convulxin Involve Glycoprotein VI and Integrin α2β1 J. Biol. Chem.1997; 272: 27035-27041).

Platelet Aggregation Measurements

A turbidimetric method was applied to measure platelet aggregation,using a Dual channel-Aggregometer (560 Ca, 230 VAC Chronolog-corp,Havertown, USA (Dikshit M, Kumari R, Srimal R C. Pulmonarythromboembolism induced alterations in nitric oxide release from ratcirculating neutrophils. The Journal of Pharmacology and ExperimentalTherapeutics 1993; 265:1369-1373). Platelet rich plasma (1×10⁸platelets/ml, 0.45 ml) was pre-warmed to 37° C. for 2 min, thenincubated with compound (3-100 μM) or an isovolumetric solvent control(0.5% DMSO) for 5 min before addition of the agonists (i.e., 2 μg/mlCollagen, 10 μM ADP, 1 U/ml Thrombin, 15 ng/ml Convulxin, 25 μM TRAP,1.5 mg/ml Ristocetin). The reaction was allowed to proceed for at least5 min, and the extent of aggregation was expressed in light-transmissionunits (Ivandic B T, Schlick P, Staritz P, Kurz K, Katas H A, GiannitsisE. Determination of clopidogrel resistance by whole blood plateletaggregometry and inhibitors of the P2Y12 receptor. Clinical Chemistry2006; 52:383-88).

The percentage of aggregation was calculated by using following formula:Percent Aggregation=A/B×100

Where, A is the number of division traversed by the label on chart inthe presence of Inducer & B is the total number of divisions (80).

Platelet Aggregation Studies (FIG. 3)

a) Platelet Aggregation (Human, In Vitro) (FIG. 3 a (i) (ii) (iii))

The invention including compound 1, compound 6 & 7 were found to beactive against collagen induced aggregation in vitro in human plateletrich plasma.

The in vitro anti-aggregatory activity was comparable among all thethree compounds as they inhibited collagen mediated effects at similarconcentrations.

The invention Compound 6 and 7 did not exhibit any effect on ADP andthrombin mimetic up to 300 μM, SFLLRN (TRAP) induced plateletaggregation, while Compound 1 exhibited a significant inhibition againstTRAP induced platelet aggregation even at 100 μM, thereby displaying itsnon-specific mode of action.

The compounds had no inhibitory effect against platelet aggregationinduced by another GP VI agonist, Convulxin and GP 1b specific agonist,Ristocetin.

Inference form FIG. 3 a could be drawn as that the compound 1, 6 and 7are active against collagen induced aggregation in vitro in humanplatelet rich plasma while no effect on ADP induced plateletaggregation. Moreover the compound 6 and 7 are more specific thancompound 1 which exhibits moderate efficacy against thrombin mimetic,SFLLRN (TRAP) induced platelet aggregation. The invention further had noinhibitory effect on platelet aggregation induced by GP VI specificagonist Convulxin and GP 1b specific agonist, Ristocetin.

Platelet Aggregation (Mice, Ex Vivo) (FIG. 3 b)

The compound 6 exhibits dose dependent inhibition in plateletaggregation (ex vivo) induced by collagen when administered to mice viaoral route

Inference could be that the compound 6 specifically inhibits collageninduced platelet aggregation.

GPVI- and α2β1-Mediated Platelet Adhesion Assay

Integrin α2β1 binding on soluble collagen depends on the presence ofMg²⁺/Ca²⁺ (Onley D. J., Knight C. G., Tuckwell D. S., Barnes M. J., andFarndale R. W. Micromolar Ca2+ concentrations are essential forMg2+-dependent binding of collagen by the integrin α2β1 in humanplatelets. J. Biol. Chem. 2000; 275: 24560-24564.) while GPVI mediatesadhesion over fibrillar collagen in absence of divalent ions (NieswandtB., Brakebusch C, Bergmeier W, Schulte V, Bouvard D, Nejad R M, LindhoutT., Heemskerk J W M, Zirngibl H and Fassler R. Glycoprotein VI but notα2β1 integrin is essential for platelet interaction with collagen. TheEMBO Journal, 2001; 20:2120-2130) and therefore the adhesion assays wereperformed in the presence or absence of these divalent cations.Ninety-six well micro titer plates were coated with insoluble equinetendon fibrillar type I collagen or soluble rat tail type I(non-fibrillar) collagen, maintained in acetate buffer (pH 4.5),(Nakamura T., Jamieson G. A., Okuma M., Kambayashi J., and Tandon N. N.Platelet Adhesion to Native Type I Collagen Fibrils: role of gpVI indivalent cation-dependent and-independent adhesion and thromboxane A2generation. J. Biol. Chem. 1998; 273: 4338-4334, Tandon N N, OckenhouseC F, Greco N J, Jamieson G A. Adhesive functions of platelet lackingGPIV (CD36). Blood 1991; 78: 2809-13). The unoccupied protein bindingsites on the wells were blocked with 5 mg/ml BSA at room temperature for1 hour, and then rinsed once in PBS. Washed platelets, suspended in theTyrode's-HEPES buffer (136.7 mM NaCl, 13.8 mM NaHCO₃, 0.36 mMNaH₂PO₄.H₂O, 2.6 mM KCl, 1.0 mM MgCl₂.6H₂O, 5.5 mM glucose, 0.5% BSA, pH7.4) at 1×10⁸ cells/mL were pre-incubated with compounds for 30 min at37° C. and added to the wells (10⁷/well) for 1 h at room temperature.Divalent cation-free adhesion buffer was made by replacing Mg²⁺ (1 mM)in the Tyrode-HEPES buffer with 50 μM EDTA (Yoshida S., Sudo T., NiimiM., Tao L., Sun B., Kambayashi J., Watanabe H., Luo E., and Matsuoka H.Inhibition of collagen-induced platelet aggregation by anophelineantiplatelet protein, a saliva protein from a malaria vector mosquito.Blood 2007; 111: 2007-201). After three washes in Tyrode's buffer, thenumber of adherent platelets was evaluated colorimetrically (asdescribed by Bellavite P., Andrioli G., Gizzo P., Arigliano P.,Chirumbolo S., Manzato F., Santonastaso C. A colorimetric method for themeasurement of platelet adhesion in microtiter plates. Anal. Biochem.1994; 216:444-450. Briefly, 150 μl of a 0.1 M citrate buffer (pH 5.4),containing 5 mM p-nitrophenyl phosphate and 0.1% TritonX-100 was addedto the wells after washing. After incubation for 60 min at 25° C. in theabsence of ambient light, colour was developed by the addition of 100 μlof 2N NaOH and the absorbance at 405 nm was read using a microplatereader (Powerware XS, Biotek, USA).

Platelet Adhesion Studies (FIG. 4 a)

GPVI- and α2β1-Mediated Platelet Adhesion Assay on Collagen (Human,In-Vitro)

1) The Compounds 6 and 7 partially inhibited both fibrillar and solublecollagen mediated human platelet adhesion over collagen (in vitro) whilethe effect seemed to be more prominent against fibrillar collagenmediated pathway.

Inference could be drawn as that the compound 6 is better than compound7 in inhibiting platelet adhesion over collagen coated surface.

Platelet Adhesion (Mice, Ex-Vivo) (FIG. 4 b)

The compound 6 exhibits dose dependent inhibition in platelet adhesion(ex vivo) over collagen surface when administered to mice via oralroute.

However aspirin did not exhibit any inhibitory effect on plateletadhesion over collagen coating

Inference could be drawn as that the compound 6 exhibits dose dependentreduction in platelet adhesion over collagen matrix and comparativelybetter than aspirin.

Measurement of Intracellular Calcium Concentration

Ca⁺² concentration ([Ca⁺²]i) were monitored by Fura-2AM fluorescence inthe platelets (Fowler C J, Tiger G Calibration of Fura-2 signalsintroduces errors into measurement of thrombin-stimulated calciummobilization in human platelets. Clinica Chimica Acta. 1997;265:247-261). Human PRP was incubated with 5 μM Fura-2 AM (Sigma) for 60minutes at 37° C. After 2 washes with the wash buffer, the plateletswere resuspended at 2×10⁸ cells/mL with the Tyrode's-HEPES buffer. Afterincubating with various concentrations of compound for 5 minutes, theplatelets were stimulated with collagen (5 μg/mL) (the concentration ofcollagen was varied in accordance with cell number). The Fura-2fluorescence was measured for 5 minutes at an excitation wavelength of340/380 nm and emission wavelength of 500 nm (Yoshida S., Sudo T., NiimiM., Tao L., Sun B., Kambayashi J., Watanabe H., Luo E., and Matsuoka H.Inhibition of collagen-induced platelet aggregation by anophelineantiplatelet protein, a saliva protein from a malaria vector mosquito.Blood 2007; 111: 2007-201) using a fluorescence spectrophotometer(VARIAN, Cary Eclipse).

Measurement of Intracellular Calcium Concentration (FIG. 8)

Since an increase in [Ca²⁺]i is considered to play a pivotal role inplatelet aggregation, the possible involvement of Compound 6 in theregulation of [Ca²⁺]i was investigated. The compound 6 exhibited aconcentration dependent inhibition in enhanced calcium mobilizationduring collagen stimulation.

Inference could be drawn as that the compound 6 inhibited [Ca²⁺]imobilization in a concentration dependent manner.

Immunoblotting

Platelet rich plasma (2×10⁸/ml) was pre-incubated with test compound(10-100 μM) or the isovolumetric solvent control (0.5% DMSO) for 5minutes, followed by the addition of collagen, to trigger plateletactivation. The reaction was stopped by the addition of ice cold stopbuffer (5 mM EDTA, 5 mM EGTA) and the suspensions were centrifuged at800×g for 10 minutes and then immediately resuspended in 300 μl of lysisbuffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 10 μg/mlaprotinin, 1 mM PMSF, 10 μg/ml leupeptin, 10 mM NaF, 1 mM sodiumorthovanadate, and 5 mM sodium pyrophosphate) (Ezumi Y, Shindoh K, TsujiM, and Takayama H Physical and Functional Association of the Src FamilyKinases Fyn and Lyn with the Collagen Receptor Glycoprotein VI-FcReceptorg Chain Complex on Human Platelets. J. Exp. Med. 1998; 188:267-276). Lysates were centrifuged at 12,000×g for 5 minutes, afterwhich the supernatants were dissolved in Laemmeli sample buffer(Laemmeli, 1970). Samples containing 30 μg of protein were separated on8% SDS-PAGE; the proteins were electrotransferred to a nitrocellulosemembrane by semi-dry method (Amersham Biosciences). The membranes wereblocked with TBST (10 mM Tris-base, 100 mM NaCl, and 0.01% Tween 20)containing 5% BSA overnight at 4° C., then probed with following primaryantibodies for 2 h: anti-p-Tyr (PY20:4 G10-1:1) (diluted 1:10000 inTBST) or β-Actin (1:5000). Membranes were washed for 30 minutes and thenincubated with horseradish peroxidase-linked anti-mouse IgG (diluted1:20000 in TBST) and anti-Rabbit IgG (1:5000) for 1 h. Immunoreactivebands were detected by chemiluminescence using the ECL-enhancedchemiluminescence system (Inoue K, Ozaki Y, Satoh K, Wu Y, Yatomi Y,Shin Y and Morita T Signal Transduction Pathways Mediated byGlycoprotein Ia/IIa in Human Platelets: Comparison with those ofGlycoprotein VI. Biochem. I and Biophys. Res. Comm. 1999; 256: 114-120).

Immunoblotting (FIG. 9)

-   1) The activation of platelets by collagen contributes to the    assembly and stabilization of various signaling complexes. This    involves tyrosine phosphorylation of various proteins in platelets.-   2) The compound 6 attenuated the tyrosine phosphorylation of various    proteins in collagen stimulated platelets in a concentration    dependent manner.

Inference could be drawn as that the compound 6 inhibited the tyrosinephosphorylation of platelet proteins following collagen stimulation.

Amidolytic Assay of Thrombin Activity (FIG. 5)

In previous experiments, the compound 1 exhibited a significantinhibitory effect on thrombin amidolytic activity at 300 μM and 1 mMafter 15 min of substrate incubation. The compound 7 actively attenuatedthe proteolytic cleavage of substrate by thrombin in a concentrationdependent manner. The compound 6 in contrast, remained ineffective anddid not exhibit any thrombin inhibitory action or concentrationdependency.

Inference could drawn as that the compound 1 and 7 non-specificallyattenuated thrombin amidolytic activity while compound 6 did not exhibitany of such non-specific thrombin inhibitory property.

Animal Model Studies (Ex Vivo/In Vivo Models)

The animals were obtained from the National Laboratory Animal Centre ofCentral Drug Research Institute, Lucknow. All the animal experimentswere subjected to Institutional Animal Ethical Committee (IAEC)guidelines and were conducted according to the guidelines ofExperimental Animal Care issued by the Committee for Purpose of Controland Supervision of Experiments on Animals (CPCSEA). The animals werehoused in polypropylene cages and maintained on standard chow diet andwater ad libitum and on 12 hr/12 hr light-dark cycle at temperature:25±2° C., humidity: 45-55% and ventilation: 10-12 exchanges/hr.

Collagen Epinephrine Induced Pulmonary Thromboembolism:

To assess the antithrombotic efficacy of Compounds, mice were dividedinto vehicle, aspirin and Compounds treated groups, and each groupincluded ten animals. Pulmonary thromboembolism was induced by injectinga mixture of collagen (150 μg/ml) and adrenaline (50 μg/ml) into thetail vein to achieve final doses of collagen (1.5 mg/kg) and adrenaline(0.5 mg/kg) to induce hind limb paralysis or death. Results have beenreported as percentage protection, which represents protection againstcollagen and epinephrine induced thrombosis and expressed as:Percent Protection=(1−(P _(test) /P _(control)))×100

P_(test)—number of animals paralyzed/dead in test compound-treatedgroup;

P_(control)—number of animals paralyzed/dead in vehicle treated group.

Collagen Epinephrine Induced Pulmonary Thromboembolism (FIG. 1):

a) The compound 6 (30 μM/Kg) displayed a remarkable antithromboticefficacy (1 hr p.o dosing) which sustained for more than 24 hours andthus highlights its excellent bioavailability.

b) The compound 6 exhibited significant antithrombotic efficacy even upto 24 hours of its administration. Although the Compound 1 exhibitedalmost similar efficacy like compound 6, but only up to 12 hours afterwhich it gradually becomes ineffective, while compound 7 displayedrelatively weaker antithrombotic potential throughout which wasmaintained for up to 12 hours, after which it also declined.

c) The standard anti-platelet drugs aspirin (170 μM/kg) and clopidogrel(70 μM/kg) were effective only up to 5 hours after which their effectperishes and that too at a very high dose sufficient enough to causebleeding complications.

Bleeding Time

Bleeding time in mice was evaluated by the method of Dejana et al,(Dejana E, Callioni A, Quintana A, Gaetano G. Bleeding time inlaboratory animals. II—A comparison of different assay conditions inrats. Thromb Res. 1979; 15:191-7). The tail 2 mm from tip of mice wasincised and the blood oozed was soaked on a filter paper, which wasmonitored at an interval of 10-15 sec till the bleeding stops. The timeelapsed from the tip incision to the stoppage of bleeding was determinedas the bleeding time. The preferred compounds, aspirin (30 mg/kg) orvehicle was given orally 60 min prior to the tail incision in a group of5 mice each.

Bleeding Time in Mice (FIG. 2)

An ideal antithrombotic drug is expected to maintain the precariousbalance between prevention of thrombosis and leaving haemostasissufficiently intact to prevent haemorrhage.

-   a) Bleeding time was found to be significantly prolonged in standard    anti-platelet drugs, aspirin (170 μM/kg) and clopidogrel (30 μM/kg)    treated mice, at the dose most effective to prevent thrombosis.-   b) The compound 1, 6 and 7 (30 μM/kg, 1 hr p.o. dosing) exhibited a    mild prolongation in bleeding time (approximately 1.5 fold) which    was significantly less as compared to the standard anti-platelet    drug, aspirin and clopidogrel.

Inference from FIG. 2 could be drawn as that at equal efficacy, Compound6 is comparatively safer than standard drug Aspirin and clopidogrel.

FeCl₃ Induced Thrombosis in Rats:

Male SD rats were anesthetized by urethane (1.25 g/kg, i.p.). Thecarotid artery was carefully dissected and a pulsed Doppler Probe(DBF-120A-CPx, CBI-8000, Crystal Biotech, USA) was placed around it torecord the blood flow velocity and patency of the blood vessels. Thecarotid artery thrombosis was induced by FeCl₃ as follows: a square (1×1mm) of Whatman Chromatography paper was immersed in 20% FeCl₃ solutionfor 5 min and placed on the carotid artery as described earlier (Kurz KD, Main B W, Sandusky G E. Rat model of arterial thrombosis induced byferric chloride. Thromb Res 1990; 60(4):269-80). Thrombosis wasmonitored as the reduction in carotid artery blood flow. The time atwhich the blood-flow velocity was decreased to zero was recorded as thetotal of occlusion (TTO) of the carotid artery. When the blood flowvelocity did not occlude within 120 minutes the time to thromboticocclusion was assigned a value of >120 minutes. (Ferric Chloride InducedThrombosis (FIG. 7))

FeCl₃ Induced Thrombosis in Rats (FIG. 7 a):

-   1. The test compound 6 significantly prolonged the time to occlude    the carotid artery in rats, thus displaying its remarkable    antithrombotic potential, while Compound 1 failed to exhibit any    protection at the same dose (FIG. 7 a (i)).-   2. The standard anti-platelet drug aspirin, even at a very high    dose, remained ineffective in prolonging the time to occlusion in    the same.-   3. The COMPOUND 6 (10, 30 & 100 μMl/kg) dose dependently exhibited    prolongation in time to occlusion compared to normal rats with    significant elevation at 30 & 100 μM/kg in rats (FIG. 7 a (ii)).

Inference could be made to the fact that the compound 1 is ineffectivewhile compound 6 exhibits significant protection and is thus better.

Arterio-Venous Shunt Model in Rats

Rats were grouped into control, aspirin and test compound group, eachgroup having six animals. Rats were anesthetized with urethane (1.25g/kg). Cervical incision was made and carotid artery and its contralateral jugular vein was exposed to prepare a shunt by usingpolyethylene tubes. Two 7 cm siliconized polyethylene tubes (0.5/1.0 mminner/outer diameter) were linked to a central 6 cm silicon tube(1.0/1.5 mm, inner/outer diameter) containing a 5 cm silk thread(pre-weighed) and were filled with saline. The shunt assembly wascannulated between the jugular vein and contra-lateral carotid arteryand blood was allowed to circulate through the shunt. Blood flow throughthe shunt was maintained for 10 minutes, subsequently the central partof the shunt was removed and silk thread having thrombus deposit wastaken out and weighed. The thrombus adhered/deposited on thread wascalculated by subtracting the wet weight of the silk thread. Thestandard drugs and test compounds were given 1 hr prior to theestablishment of arterio-venous shunt (Tohti I, Tursun M, Umar A, TurdiS, Imin H, Moore N. Aqueous extracts of Ocimum basilicum L. (sweetbasil) decrease platelet aggregation induced by ADP and thrombin invitro and rats arterio-venous shunt thrombosis in vivo. Thrombosis Res.2006; 118: 733-739).

Arterio-Venous Shunt Model in Rats (FIG. 6 a)

The compound 6 and 7 were almost equally effective in reducing thethrombus weight significantly and their efficacy was comparable to thoseof standard anti-platelet drugs, aspirin and clopidogrel in rats.

The inference could be made as that the antithrombotic efficacy of bothcompound 6 and 7 is identical.

Hyperlipidemic Hamster Model

Golden Syrian hamster (100-120 g) obtained from the National LaboratoryAnimal Centre of Central Drug Research Institute, Lucknow was used inthis study. All the procedures involving hamsters were subject toInstitutional Animal Ethical Committee (IAEC) guidelines. The animalswere housed in polypropylene cages and maintained on standard chow dietand water ad libitum and on 12 hr/12 hr light-dark cycle at temperature25±2° C., humidity 45-55% and ventilation: 10-12 exchange/hr. Animalswere randomly divided into one of two groups, Normal chow fed (Protein186.2 g/kg, fat 62.5 g/kg, fibre 45.3 g/kg, Nitrogen free extract 537.7g/kg, Vitamins, Minerals), and HCHF fed (Chow diet supplemented with 3%Cholesterol and 15% Saturated fat) for three months (n=16). These groupswere again divided into those receiving aspirin (30 μM) or vehicle (ingum acacia suspension) or one of the preferred compounds (Example-1 orExample-15) (30 μM/Kg). After the twelve-week feeding period, theanimals were fasted for 14 hours prior to sacrifice (Cheema S K, CornishM L., Bio F1B hamster: a unique animal model with reduced lipoproteinlipase activity to investigate nutrient mediated regulation oflipoprotein metabolism. Nutrition & Metabolism 2007, 4:27). The hamsterswere anaesthetized by anesthetic ether sniffing, heart was punctured andfasting blood samples were collected in tubes containing 2.5% tri sodiumcitrate. Some amount of Whole blood was used for aggregation studies andremaining whole blood was centrifuged at 180 g for ten minutes forseparation of platelet rich plasma (PRP). 10% ACD (39 mM citric acid, 75mM tri-sodium citrate, 135 mM D-glucose, pH 4.5) was added to Plateletrich plasma and was spun at 800 g for 10 min to get platelet pellet andplatelet poor plasma (PPP). Platelet poor plasma was used forcoagulation studies and while platelets were used to assess adhesionover collagen coated surface.

Platelet Adhesion on Collagen Coated Surface (Hyperlipidemic Hamsters)

Platelet adhesion to collagen was measured in polystyrene 96-well microtiter plates. Micro titer plates were coated with insoluble equinetendon native fibrillar type I collagen (Chrono-Log Corp. Havertown,USA), 2 μg/well in 5 mM acetic acid (Tandon N N, Ockenhouse C F, Greco NJ, Jamieson G A. Adhesive functions of platelet lacking GPIV (CD36).Blood 1991; 78: 2809-13) overnight at 4° C. The unoccupied proteinbinding sites on the wells were blocked with 0.5% BSA in Tyrode's-HEPESbuffer (136.7 mM NaCl, 13.8 mM NaHCO₃, 0.36 mM NaH₂PO₄.H₂O, 2.6 mM KCl,1.0 mM MgCl₂.6H₂O, 5.5 mM glucose, pH 7.4) at room temperature for 1hour, and then rinsed once with PBS. Washed platelets were suspended inthe Tyrode's-HEPES buffer and added to the wells (2×10⁷ cells/well) for1 h at room temperature. After three washes with PBS, the number ofadherent platelets was evaluated colorimetrically (Bellavite P.,Andrioli G., Gizzo P., Arigliano P., Chirumbolo S., Manzato F.,Santonastaso C. A colorimetric method for the measurement of plateletadhesion in microtiter plates. Anal. Biochem. 1994; 216:444-450).Briefly, 150 μl of a 0.1 M citrate buffer (pH 5.4), containing 5 mMp-nitrophenyl phosphate and 0.1% TritonX-100 was added to the wellsafter washing. After incubation for 30 minutes at 25° C. in the absenceof ambient light, colour was developed by the addition of 100 μl of 2NNaOH and the absorbance at 405 nm was read using a microplate reader(Powerware XS, Biotek, USA).

Platelet Adhesion on Collagen Coated Surface (Hyperlipidemic HamsterModel, Ex-Vivo) (FIG. 4 c)

-   1) The standard anti-platelet drug aspirin displayed no alteration    in platelet adhesion while clopidogrel exhibited a significant    reduction in platelet adhesion over collagen coated surface.-   2) The compound 6 but not 7 exhibited a significant inhibition in    platelet adhesion over collagen surface in hyperlipidemic hamster    model of atherosclerosis, with a better efficacy profile than    aspirin.-   3) Further the compound 6 exhibited a dose dependent inhibition in    platelet adhesion over collagen coated surface.

The inference could be drawn as that the compound 6 exhibits significantattenuation in platelet adhesion over collagen surface in contrast tocompound 7 which exhibits no effect on the same.

Whole Blood Aggregation

Whole blood aggregation studies were performed on a dual channelaggregometer (560 Ca, 230 VAC, Chrono-log Corp, USA) using the impedancemethod (Ivandic et al, 2006). The citrated whole blood (450 μl) weremixed by gentle inversion then diluted with physiological saline (0.9%NaCl, 450 μl) and calcium chloride (10 μl, 1.0% w/v), were incubated at(37° C.) until the time of use. Aggregation response was taken afteradding inducers at the following final concentrations: ADP 10 μM;Thrombin 1 U/ml, Collagen 2.5 μg/ml, with stirring. After inducersaddition, the impedance was measured over a time interval of 6 minutes.Platelet aggregation was measured by calculating impedance (Ohms). Thepercentage of aggregation was calculated by conventional method asdescribed earlier (Torres Duarte A P, Dong Q S, Young J, Abi-Younes S,Myers A K. Inhibition of platelet aggregation in whole blood by alcohol.Thromb Res 1995; 78 (2):107-15, Kumari R, Singh M P, Seth P, Dikshit M.Inhibition of platelet aggregation by a protein factor present in ratperipheral polymorphonuclear leukocyte supernatant. Thromb Res 1998; 91(2):75-82).

Whole Blood Aggregation in Hyperlipidemic Hamsters (Ex Vivo) (FIG. 3 c)

-   1) The mean maximal platelet aggregation in response to ADP,    collagen and thrombin was found to be significantly increased in    high cholesterol high fat diet fed hamsters compared with those on    normal chow.-   2) Aspirin (100 μM/kg) treated HCHF hamsters displayed reduced    platelet aggregation in response to collagen but not in ADP and    thrombin mediated effects.-   3) Clopidogrel (30 μM/kg) treatment also inhibited ADP and collagen    induced platelet aggregation but not thrombin mediated platelet    responses.-   4) The invention (Example-6) significantly inhibited collagen    induced platelet Aggregation in high fat group in dose dependent    manner while example 7 was unable to do so. Also ADP and thrombin    induced activation remained unhindered.

Inference could be drawn as that the Compound 6 is better than Compound7 in attenuating collagen stimulated platelet aggregation inhyperlipidemic hamsters.

FeCl₃ Induced Thrombosis in Hyperlipidemic Hamsters

HCHF treated hamsters were anesthetized by urethane (1.25 g/kg, i.p.).The carotid artery was carefully dissected and a pulsed Doppler Probe(DBF-120A-CPx, CBI-8000, Crystal Biotech, USA) was placed around it torecord the blood flow velocity and potency of the blood vessels. Thecarotid artery thrombosis was induced by FeCl₃ as follows: a square (1×1mm) of Whatman Chromatography paper was immersed in 30% FeCl₃ solutionfor 5 min and placed on the carotid artery as described earlier (Kurz KD, Main B W, Sandusky G E. Rat model of arterial thrombosis induced byferric chloride. Thromb Res 1990; 60(4):269-80). Thrombosis wasmonitored as the reduction in carotid artery blood flow. The time atwhich the blood-flow velocity was decreased to zero was recorded as thetotal of occlusion of the vessel.

b) FeCl₃ Induced Thrombosis in Hyperlipidemic Hamsters (FIG. 7 b):

-   1) High fat diet treatment reduced the time to occlude the carotid    artery, thus displaying the incidence of a pro-thrombotic state    during hyperlipidemia in hamsters.-   2) The standard antithrombotic drugs aspirin and clopidogrel    significantly prolonged the time to occlusion (TTO) in hamsters.-   3) The compound 6 significantly increased the time to occlusion in    hamsters, thus confirming its substantial antithrombotic efficacy in    a disease model, while compound 1 and compound 7 remained    ineffective and thus did not display antithrombotic characteristic    in the same.

Inference could be made to the fact that the compound 1 and 7 wereineffective while compound 6 significantly prolonged the time toocclusion and is therefore better than the two.

Arterio-Venous Shunt Model in Hyperlipidemic Hamsters (FIG. 6 b):

-   1) High fat diet treatment significantly increased the thrombus    weight, thus displaying a pro-thrombotic state induced by    hyperlipidemic diet mimicking the clinical condition.-   2) The standard antithrombotic drugs aspirin and clopidogrel    significantly reduced the weight of thrombus formed.-   3) The Compound 6 significantly reduced the thrombus weight even at    a very low dose (10 μM/kg) and was almost equivalent to those of    standard drugs.-   4) The inference could be made as that the compound 6 significantly    reduces thrombus weight even at 10 μM/kg and is better.

Pharmaco-Kinetic Data (FIG. 10)

In-vivo pharmacokinetic parameters were generated in male NZ rabbits ata dose of 20 mg/kg by oral route of administration. The blood sampleswere drawn at 0.25 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 12, 18 and 24 hrpost dose, processed and analyzed by Liquid Chromatography-MassSpectrometry (LC-MS). Plasma concentration at different time points wereplotted to give Plasma Concentration Time Profile and the data wasfitted non-compartmentally using Win NonLin 5.1 version software and theestimates of pharmacokinetic parameters were derived with SD meanvalues. Maximum plasma concentration (C_(max)), time to achieve maximumplasma concentration (t_(max)), Area under curve (AUC) and meanresidence time (MRT) of Compound 1, Compound 6 and Compound 7 are givenin Table-1.

TABLE 1 represents estimates of pharmacokinetic parameters of 6 and 7 inmale NZ rabbits at a dose of 20 mg/kg body weight Compound 1(a) 1:1 PKMixture Parameters of 6 & 7 Compound 6 Compound 7 Cmax 947.02 ± 237.4149.49 ± 53.12  112.40 ± 10.25 (ng/ml) Tmax  0.68 ± 0.29 0.75 ± 0.144 8.67 ± 0.66 (h) AUC 6524.15 ± 1502.7 874.8 ± 232.01 1112.81 ± 169.77(ng · h/ml) MRT (h) 16.20 ± 1.46 5.25 ± 1.75  16.20 ± 1.46

Summary of the Biological Assays

TABLE 2 Summary of the biological assays 1:1 mixture Test systems of 6 &7 Compound 6 Compound 7 Inference Collagen-epinephrine Up to 18 hrs >24hrs <12 hrs Compound 6 more active, induced thrombosis Effect is longlasting in mice Bleeding Time in 2 fold 1.5 fold 1.5 fold At equalefficacy Compound 6 mice is more safe Arterio-venous shunt ProtectiveProtective Protective Similar activity model in rat Arterio-venous shuntProtective Compound 6 is better model in hyper- Even at 10 μM lipidemichamsters Ferric chloride Not Protective Compound 6 is better inducedthrombosis protective in rats Ferric chloride Not Protective Notprotective Compound 6 is better induced thrombosis protective inhyper-lipidemic hamsters Collagen induced ~10 μM ~10 μM >10 μM Compound6 is better than platelet aggregation Compound 7 studies (Human) IC50TRAP induced >100 μM >500 μM >500 μM Both compound 6 & 7 are moreplatelet aggregation specific than the mixture studies (Human) IC50Platelet adhesion Effective Effective (30 Effective Compound 6 is better(100 μM) &100 μM) (100 μM) Whole blood Effective Effective No effectCompound 6 is better than aggregation in Compound 7 hyperlipidemichamsters Platelet adhesion Effective Effective No effect Compound 6 isbetter than hyperlipidemic Compound 7 hamsters

Advantages:

-   1. The compounds of the present invention act as inhibitors of    collagen induced platelet activation and adhesion-   2. The other advantage is to avoid any racemization at the    α-carboxylic center, during N-alkylation, the reagent LiHMDS at low    temperatures is claimed to furnish Methyl    N-p-methyl-phenylmethyllpyroglutamate in good chiral purity.-   3. Further advantage is to avoid any racemization at the    α-carboxylic center.-   4. The compounds of the present invention showed fast absorption    with effective C_(max) values-   5. The compounds of the present invention showed optimum levels of    solubility-   6. The compounds of the present invention showed low plasma levels-   7. The compounds of the present invention showed low peripheral    accumulation-   8. The compounds of the present invention showed lower levels of    toxicity

We claim:
 1. Isolated diastereomer compound of formula 6 or 7 havinggeneral formula1-(4-methylphenylmethyl)-5-oxo-{N-[(3-t-butoxycarbonyl-aminomethyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamides;


2. A process for the a preparation of compound of formula 6 and acompound of formula 7

wherein the process comprises the steps of: a) reacting methylpyroglutamate with p-methyl benzyl bromide in an aprotic solvent inpresence of LiHMDS at a temperature ranging between 0 to 350 C for aperiod ranging between 1 to 4 hr, quenching the reaction mixture withHCl and obtaining the compound of formula 2

b) reacting compound of formula 2 with sodium carbonate at a temperatureranging between 0° C. to 30° C. for a period ranging between 0.5 to 4 hrto obtain 2S)—N-(p-methylphenylmethyl) pyroglutamic acid of formula 3;

c) reacting the compound of formula 3 with a compound of formula4[(3R)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine] or formula5[(3S)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine]

in an aprotic solvent selected from a group consisting ofdichloromethane, tetrahydrofuran, dioxane in presence of a couplingreagent selected from the group consisting of dicyclohexylcarbodiimide,benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophophate,or an activating agent 1-hydroxy benzotrizole or isobutyl chloroformateat a temperature ranging between −20° C. to +30° C. for a period rangingfrom 1 to 3 hrs followed by purification using chromatography to producethe compound of formula 6 or 7 respectively.
 3. The process as claimedin claim 2, wherein the compound of formula 3 is reacted with oxalylchloride at 0° C. to obtain the acid chloride followed by reaction withcompound of formula 4 or 5 in presence of triethylamine indichloromethane to obtain the compound of formula 6 or 7 respectively.4. The process as claimed in claim 2, wherein the compound of formula 3is reacted with the compound of formula 4 or 5 in presence of a couplingreagent dicyclohexylcarbodiimide and 1-hydroxy benzotrizole indichloromethane at a temperature ranging between −5° C. to 0° C. for aperiod ranging between 2 h to 3 h to obtain the compound of formula 6 or7 respectively.
 5. The process as claimed in claim 2, wherein thecompound of formula 3 is reacted with compound of formula 4 or 5 in thepresence of diisopropyl ethylamine, andbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophophate,in dichloromethane at temperature ranging between −50° C. to 0° C. for aperiod ranging between 2 h to 3 h followed by stirring at temperature ata temperature ranging between 0° C. to 25° C. to obtain compound offormula 6 or 7 respectively.
 6. The process as claimed in claim 2,wherein the compound of formula 3 is reacted with compound of formula 4or 5 in presence of triethyl amine, and isobutyl chlorormate, in THF at−20° C., for a period ranging between 1 h to 2 h followed by stirringtemperature ranging between 0° C. to 250° C. to obtain the compound offormula 6 or
 7. 7. The process as claimed in claim 2 wherein thepurification of the compound of formula 6 and 7 is carried out bycrystallization using a solvent selected from the group consisting ofpentane, hexane, cyclohexane, toluene and ethyl acetate.
 8. Apharmaceutical composition comprising an antithrobotic effective amountof the compound of formula 6 or 7 with pharmaceutically acceptableadditives and excipients.
 9. The composition as claimed in claim 8wherein the pharmaceutically acceptable additives are selected from thegroup consisting of DMSO, gum acacia or CMC, beta cyclodextrin, or anyother pharmaceutically acceptable excipients.
 10. Pure diastereomercompound of formula 6,(2S)-1-(4-methylphenylmethyl)-5-oxo-(3S)—{N-[(3-t-butoxycarbonylaminomethyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamide, having thestructural formula:


11. Pure diastereomer compound of formula 7,(2S)-1-(4-methylphenylmethyl)-5-oxo-(3R)—{N-[(3-t-butoxycarbonyl-aminomethyl)]-piperidin-1-yl}-pyrrolidine-2-carboxamide,having the structural formula:


12. A process for the preparation of a compound of formula 6 wherein theprocess comprises the steps of: a) reacting methyl pyroglutamate withp-methyl benzyl bromide in an aprotic solvent in presence of LiHMDS at atemperature ranging between 0 to 35° C. for a period ranging between 1to 4 hr, quenching the reaction mixture with HCl and obtaining thecompound of formula 2

b) reacting the compound of formula 2 with sodium carbonate at atemperature ranging between 0° C. to 30° C. for a period ranging between0.5 to 4 hr to obtain 2S)—N-(p-methylphenylmethyl) pyroglutamic acid offormula 3; and,

c) reacting the compound of formula 3 with a compound of formula4[(3R)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine] or formula5[(3S)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine] in an aproticsolvent selected from a group consisting of dichloromethane,tetrahydrofuran, dioxane in presence of a coupling reagent selected fromthe group consisting of dicyclohexylcarbodiimide,benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophophate,or an activating agent 1-hydroxy benzotrizole or isobutyl chloroformateat a temperature ranging between −20° C. to +30° C. for a period rangingfrom 1 to 3 hrs followed by purification using chromatography to producecompound of formula
 6. 13. A process for the preparation of a compoundof formula 7 wherein the process comprises: a) reacting methylpyroglutamate with p-methyl benzyl bromide in an aprotic solvent inpresence of LiHMDS at a temperature ranging between 0 to 35° C. for aperiod ranging between 1 to 4 hr, quenching the reaction mixture withHCl and obtaining the compound of formula 2

b) reacting the compound of formula 2 with sodium carbonate at atemperature ranging between 0° C. to 30° C. for a period ranging between0.5 to 4 hr to obtain 2S)—N-(p-methylphenylmethyl) pyroglutamic acid offormula 3; and

c) reacting the compound of formula 3 with a compound of formula4[(3R)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine] or Formula5[(3S)—N-(t-butoxycarbonyl)-3-aminomethyl-piperidine] in an aproticsolvent selected from a group consisting of dichloromethane,tetrahydrofuran, dioxane in presence of a coupling reagent selected fromthe group consisting of dicyclohexylcarbodiimide,benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophophate,or an activating agent 1-hydroxy benzotrizole or isobutyl chloroformateat a temperature ranging between −20° C. to +30° C. for a period rangingfrom 1 to 3 hrs followed by purification using chromatography to producecompound of formula 6 or 7 respectively.